Living organisms must deal with numerous random assaults on the structural and information integrity of their DNA, and death can often result in the absence of an adequate defense. Therefore, an organism must have the flexibility to act, almost instantaneously, to protect against the loss of genetic information. Common factors that can alter DNA include man-made environmental pollutants as well as naturally occurring radiation. This proposal is focused on elucidating the biochemical basis of an important and unusual survival mechanism that enables the bacterial organism, Escherichia coli, to cope with the presence of extensive DNA damage caused by UV radiation. The phenomenon has been named "SOS-induced error prone repair." In the presence of high doses of UV radiation, damage to the DNA can result in the loss of coding information via creation of cyclobutane dimers and other types of photoadducts, and from the direct loss of DNA replication if they are not removed either through excision repair or genetic recombination. When the DNA damage is extensive enough to overload the constitutive repair pathways in the cell, then the cell responds by turning on at least 20 different genes in the SOS repair pathway. Following induction of these genes, the damaged DNA can then be replicated. However, the SOS-induced DNA repair is accompanied by a significant, 10 - 100 fold increase in mutagenesis. It is the biochemical basis of this mutagenic response that the grant is intended to address. DNA polymerases and other auxiliary proteins required to replicate DNA will be isolated and purified from SOS-induced and uninduced cells. The main object is to identify proteins that are present predominantly in SOS-induced cells that allow DNA replication to proceed past non-coding DNA template lesions. Synthetically prepared non-coding lesions will be used so that an unambiguous measurement can be made of the efficiency of nucleotide insertion and DNA chain extension at a well-defined, uniquely situated, template lesion. Proof that a particular protein is required for error prone repair will be sought by sequencing and cloning proteins able to enhance the efficiency of lesion bypass, and then construction deletion mutants to determine the effect of the gene products on SOS mutagenesis. This grant is also focused on an E. coli DNA polymerase, DNA polymerase II, whose function in the cell has remained unresolved for almost two decades. Evidence is presented that this enzyme is strongly induced as part of the SOS response to DNA damage, and has the property that it can insert and bypass at non-coding DNA lesions.